The majority of vaccines currently in development belong to a specific class of subunit vaccine compositions, which consist of recombinant or purified pathogen-specific proteins or encoded antigens (from DNA) that will be expressed and presented in vivo in order to accomplish or elicit the desired immunogenic response. This type of vaccine presents an antigen to the immune system without introducing viral particles, whole or otherwise. While evidence suggests that live, attenuated pathogens and viral vectors can induce protective effects, they often cause unwanted side effects or raise safety concerns, which is one reason why subunit vaccines have risen to prominence in the field (Arvin et al., “New viral vaccines”, Virology, 344:240-249 (2006); Yang et al., “A novel peptide isolated from phage library to substitute a complex system for a vaccine against staphylococci infection”, Vaccine, 24:1117-1123 (2006))
One weakness of this technique is that isolated proteins can be denatured and thus will be associated with antibodies that are distinct from the desired antibodies. Another method of making subunit vaccine involves extracting an antigen's gene from the targeted virus (or bacterium) and inserting this gene into another virus or attenuated bacterium to make a recombinant virus or bacteria.
Researchers have also inserted an antigen's gene derived from a targeted virus into yeast. Examples of such resultant vaccines are well known in the art, including subunit viral vaccines derived from hepatitis B surface antigen (HBsAG) produced in yeast cells (Recombivax HB from Merck).
Such subunit vaccines, when administered alone, have relatively low efficacy for immune system activation, generally exhibiting poor immunogenicity (Toes et al., “Peptide vaccination can lead to enhanced tumor growth through specific T-cell tolerance induction”, Proc. Natl. Acad. Sci., 93:7855-7860 (1996)) and thus require the addition of adjuvants in order to elicit the appropriate level of immune system response to a particular antigen, initially through the innate, then subsequently the adaptive immune system (Grasso, P et al., Essentials of Pathology for Toxicologists, CRC Press (2002)).
Preferably, the adjuvant should be able to improve or facilitate antigen uptake by antigen presenting cells (APCs) and, ideally, then induce an Ag-specific immune response while simultaneously eliciting minimal toxicity to the individual.
Currently, aluminum-based adjuvants, such as aluminum phosphate and aluminum hydroxide, dominate their field and, prior to 2009, were the only licensed adjuvants in the U.S. Unfortunately, while the use of adjuvants such as alum and MF59 (oil-in-water, proprietary adjuvant owned by Novartis) can augment certain response to specific Ags, such agents can sometimes lead to induction of undesired, inappropriate responses (ie. generation of a humoral response rather than a cell mediated response) (Roberts et al., “Phase 2 study of the g209-2M melanoma peptide vaccine and low-dose interleukin-2 in advanced melanoma”, J. Immunother., 29(1):95-101 (2006)).
Additionally, these inorganic adjuvants face numerous other problems as they are frost sensitive and not readily lyophilizable. The limitations placed on vaccines by adjuvants that are not freeze-compatible severely restrict the use of such vaccines and make them unavailable in many areas in the world. In fact, liquid formulations of Al-based vaccines against diphtheria, pertussis, tetanus, hepatitis B and influenza (type B) should not be frozen (Kartoglu et al., “Validation of the shake test for detecting freeze damage to adsorbed vaccines”, Bull World Health Organ., 88:624-631 (2010)). Unfortunately, practices that expose a wide variety of vaccines to sub-zero temperatures are widespread, in both developed and developing countries, across all levels of the respective health systems (Matthias et al., “Freezing temperatures in the vaccine cold chain: a systematic literature review”, Vaccine, 25:691-697 (2007)).
When a vaccine is damaged by freezing, the potency lost can never be restored, resulting in permanent damage to the underlying composition itself. As a result, freeze-damaged vaccines have lower immunogenicity and are more likely to cause local reactions, such as sterile abscesses (Dimayuga et al., “Effects of freezing on DTP and DTP-IPV vaccines, adsorbed”, Can. Commun. Dis. Rep., 21:101-103 (1995); Mansoor et al., “Vaccine adverse events reported in New Zealand”, NZ Med. J., 110:270-272 (1997)). Accordingly, much has been devoted to overcoming the inherent problems within the cold chain relative to vaccine compositions.
The cold-chain, a supply chain for pharmaceutical drugs based on temperature control, is a laborious process that attempts to keep vaccines at the suggested 2-8° C. range, and thus costs companies and organizations (ie. UNICEF) millions of dollars every year. Freeze-sensitive vaccines represent over 30% of the $439 million UNICEF spent on all vaccines in 2005 and the $757 million spent in 2010. Carrying-containers using ice (prominent in developing countries), defective refrigerators, and extreme cold climates can impel these vaccines to freeze and render them ineffective. Rate of exposure to freezing temperatures in developed and developing countries is 13.5% and 21.9%, respectively—making this a global concern. Freezing is a risk at any level of the cold chain, and serves as a major problem for many salient vaccines.
In the face of the above mentioned limitations with respect to aluminum-based adjuvants in vaccine compositions, it has been shown that liposomes may be a viable alternative as an adjuvant providing similar immunogenicity, without the problems associated with freeze sensitivity and lyophilization. Furthermore, these freeze sensitive adjuvants found in the prior art have also failed to elicit adequate immune responses in many cases and, often times, do not bind effectively to all protein antigens. This has spurred interest in other forms of adjuvants which may be more versatile and without the encumbrances identified in Al-based adjuvants.
In the approximately 1,400 publications about liposomal vaccines since 1974, over 25% have been published in the past three years, propelling the creation of multiple vaccines using liposomal adjuvants against influenza (Inflexal®V) and hepatitis (Epaxal®). Both of these vaccines must be stored at 2-8° C. and should not be frozen. Other liposomal vaccines that are currently moving toward regulatory approval are based on synthetic, cationic lipids which are insufficiently immunogenic, and are thus often combined with immunostimulators such as lipid A. Similar studies have also proven an effective composition in other alternative, bioactive lipid based vaccine compositions using Lipid A (Coler et al., “Development and Characterization of Synthetic Glucopyranosyl Lipid Adjuvant System as a Vaccine Adjuvant”, PLoS One, 6:e16333 (2011)).
Liposomes are lipid-bilayer, vesicular structures within which a variety of substances may be entrapped and delivered in vivo in a safe and effective manner. Liposomes are composed largely of natural or synthetic phospholipids which, over the last several decades, have been utilized for effective delivery of therapeutic agents ranging from enzyme replacement therapy (Jain et al., “Muco-adhesive multivesicular liposomes as an effective carrier for transmucosal insulin delivery”, J. Drug Target, 15:417-427 (2007)), to intracellular delivery of chelating agents in cases of heavy metal poisoning (Rahman et al., “Preparation and prolonged tissue retention of liposome-encapsulated chelating agents”, J. Lab. Clin. Med., 83:640-647 (1974)), to even possible treatments for certain cancers (Gregoriadis et al., “Drug-carrier potential of liposomes in cancer chemotherapy”, Lancet, 1:1313-1316 (1974)).
More recently, liposomes have been found to be suitable vaccine adjuvants in having the ability to prevent antigen degradation while enhancing its uptake by APCs (Gregoriadis et al., “The immunological adjuvant and vaccine carrier properties of liposomes”, J. Drug Target, 2:351-356 (1994); Brunel et al., “Cationic lipid DC-Chol induces an improved and balanced immunity able to overcome the unresponsiveness to the hepatitis B vaccine”, Vaccine, 17:2192-2203 (1999)).
Liposomes have been considered as useful vehicles for the containment of particular antigens, though the choice of lipid used in the synthesis of liposomes greatly impacts their physico-chemical and immunogenic properties. Much research has been devoted to the use of many diverse lipids with the aim of refining the adjuvanting effect of liposome-delivered vaccines (Gluck, R., “Liposomal presentation of antigens for human vaccines”, Vaccine Design: The Subunit and Adjuvant Approach., 347-361 (1995)). Phospholipid molecules, in particular, have been examined for their distinctive regions of non-polar (comprised of one of more fatty acid chains or cholesterol) and polar (consisting of a phosphate group linked to tertiary or quarternary ammonium salts). The polar region can have a net negative (anionic), neutral or positive (cationic) surface charge, which directly impacts the specific behavior and function of the specific liposome (Milicic et al., “Small cationic DDA:TDB liposomes as protein vaccine adjuvants obviate the need for TLR agonists in inducing cellular and humoral responses”, PLoS ONE, 7(3):1-10 (2012)).
While the potential of antigens entrapped in liposomes for use as potential vaccines have shown promising results, there are a great many alternatives to preferred liposomal compositions, particularly with respect to entrapment of the antigens. For instance, antigens to be delivered may either be entrapped within the aqueous compartment of the liposomes, incorporated into the lipid bilayer membrane (hydrophobic antigens) or adsorbed into the liposomal surface through covalent or charge-dependent, electrostatic interaction (Taneichi et al., “Induction of differential T-cell epitope by plain- and liposome-coupled antigen”, Bioconjug. Chem., 17:899-904 (2006)). More recently, strong evidence has indicated great potential for enhancing immunogenicity of cationic liposomes through addition of toll-like receptor (TLR) agonists (Bal et al., “Co-encapsulation of antigen and toll-like receipt ligand in cationic liposomes affects the quality of the immune response in mice after intradermal vaccination”, Vaccine, 29:1045-1052 (2011)). Similarly, liposomal encapsulation of CpG oligonucleotides has been shown to enhance and prolong innate system stimulation and advanced the CpG-induced immune protection against Listeria (Gursel et al., “Sterically stabilized cationic liposomes improve the uptake and immunostimulatory activity of CpG oligonucleotides”, J. Immunol., 167:3324-3328 (2001)).
Methods of manufacturing the antigen loaded liposomes of the prior art consist primarily of embodiments described in the literature involving reverse phase evaporation and related techniques (Sazoka et al., “Rapid separation of low molecular weight solute from liposomes without dilution”, Proc. Natl. Acad. Sci., 75:4194 (1978)). With respect to liposomal compositions consisting of glucopyranosyl lipid adjuvant (GLA), the methods used to develop such adjuvants are formulated as aqueous suspensions and have been used in the past to characterize the usefulness of synthetic TLR4 agonists (Anderson et al., “Physicochemical characterization and biological activity of synthetic TLR4 agonist formulations”, Colloids Surf. B. Biointerfaces, 75:123-132 (2010)). However, many of the known methods of making liposomal vaccines in the prior art have encountered several difficulties, ranging from error in antigen binding to nonspecific effects relating to the aqueous suspension. Most importantly, such vaccine formulations still suffer from the general defect found in the Al-based vaccines relating to freeze sensitivity. Thus, there remains a great need in the art to solve the problem of generating a vaccine composition capable of eliciting a consistent immunogenic response, with a high degree of selectivity and efficacy, with such activity not being diminished in the presence of sub-zero temperatures.